U.S. patent application number 12/446550 was filed with the patent office on 2010-12-16 for liposome preparation by single-pass process.
This patent application is currently assigned to MediGene AG. Invention is credited to Klaus Drexler, Heinrich Haas, Michael Wiggenhorn, Gerhard Winter.
Application Number | 20100316696 12/446550 |
Document ID | / |
Family ID | 37898375 |
Filed Date | 2010-12-16 |
United States Patent
Application |
20100316696 |
Kind Code |
A1 |
Wiggenhorn; Michael ; et
al. |
December 16, 2010 |
LIPOSOME PREPARATION BY SINGLE-PASS PROCESS
Abstract
The present invention relates to a method of preparing liposomes
in a single-pass mode. The method comprises the extusion of a
solution or suspension through a porous device and subsequently
passing said suspension or solution through a nozzle. Passing the
suspension through said nozzle may result in an atomisation of the
suspension or solution into droplets which might be employed in a
subsequent spray-drying or spray-freezing process.
Inventors: |
Wiggenhorn; Michael;
(Munchen, DE) ; Winter; Gerhard; (Penzberg,
DE) ; Haas; Heinrich; (Munchen, DE) ; Drexler;
Klaus; (Olching, DE) |
Correspondence
Address: |
MORGAN LEWIS & BOCKIUS LLP
1111 PENNSYLVANIA AVENUE NW
WASHINGTON
DC
20004
US
|
Assignee: |
MediGene AG
Planegg
DE
|
Family ID: |
37898375 |
Appl. No.: |
12/446550 |
Filed: |
November 2, 2007 |
PCT Filed: |
November 2, 2007 |
PCT NO: |
PCT/EP07/09531 |
371 Date: |
April 21, 2009 |
Current U.S.
Class: |
424/450 ; 264/13;
424/649; 425/6 |
Current CPC
Class: |
B01J 2/06 20130101; A61K
9/1617 20130101; A61K 9/1272 20130101; B01D 1/18 20130101; B01J
13/04 20130101; A61K 9/1277 20130101; A61P 31/12 20180101; A61P
31/10 20180101; A61P 35/00 20180101; A61P 31/04 20180101 |
Class at
Publication: |
424/450 ;
424/649; 264/13; 425/6 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 33/24 20060101 A61K033/24; B29B 9/12 20060101
B29B009/12 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 7, 2006 |
EP |
06 023 155.2 |
Claims
1. A method for the preparation of liposomes in a single-pass mode,
comprising the steps: providing a suspension or solution comprising
lipids, extruding the suspension or solution of step (a) through a
porous device, and subsequently passing said suspension or solution
of step (b) through a nozzle, and optionally collecting the
liposomes formed after step (c).
2. A method according to claim 1, wherein step (c) comprises
atomising the suspension or solution into droplets comprising
liposomes.
3. A method according to claim 1, further comprising the steps
dehydrating the liposomes of step (c) and optionally collecting the
dehydrated liposomes formed after (d), and optionally rehydrating
the dehydrated liposomes of step (d) in an aqueous medium.
4. A method according to claim 3, wherein the dehydration step (d)
is performed by spray-drying or by spray-freeze drying.
5. A method according to claim 1, wherein the suspension or
solution of step (a) comprises an aqueous medium.
6. A method according to claim 5, wherein the aqueous medium
comprises at least one further liquid constituent at least
partially miscible with water, preferably an organic solvent.
7. A method according to claim 6, wherein the organic solvent is an
alcohol or a ketone such as methanol, ethanol, propanol, butanol,
acetone, methylethylketone, DMF, DMSO or a mixture thereof,
preferably ethanol.
8. A method according to claim 1, wherein the suspension or
solution of step (a), comprises lipid particles.
9. A method according to claim 1, wherein the lipid particles of
the suspension or solution of step (a) are MLVs.
10. A method according to claim 1, wherein the particle size
distribution of the liposomes obtained after step (c) is reduced
compared to the particle size distribution of the lipid particles
of the suspension of step (a).
11. A method according to claim 1, wherein the liposomes obtained
after step (c) are substantially homogeneous.
12. A method according to claim 1, wherein the liposomes obtained
after step (c) have a polydispersity index of lower than 0.3,
preferably lower than 0.1.
13. A method according to claim 1, wherein the liposomes obtained
after step (c) have an average diameter of between about 50 nm and
about 500 nm, preferably between about 50 nm and 200 nm.
14. A method according to claim 1, wherein the liposomes obtained
after step (c) are unilamellar liposomes.
15. A method according to claim 1, wherein the suspension or
solution of step (a) has a lipid concentration of up to 400 nM.
16. A method according to claim 1, wherein the suspension or
solution of step (a) comprises at least one cationic lipid,
preferably DOTAP.
17. A method according to claim 1, wherein the suspension or
solution of step (a) and/or the liposomes obtained after step (c)
comprise at least one active compound.
18. A method according to claim 17, wherein the active compound is
a pharmaceutical active compound, e.g. an antimicrobial,
antifungal, antiviral, cytostatic or cytotoxic agent.
19. A method according to claim 17, wherein the ratio between water
and the further liquid constituent is between about 70:30 and about
40:60 (v/v), most preferably between about 80:20 and about 60:40
(v/v).
20. A method according to claim 1, wherein the suspension or
solution of step (a) comprises a hydrophilic excipient.
21. A method according to claim 1, wherein in step (b) the pressure
of the suspension or solution is about the same on both sides of
the porous device.
22. A method according to claim 1, wherein the porous device of
step (b) has a pore size of between about 50 and about 300 nm,
preferably of between about 100 and about 200 nm.
23. A method according to claim 1, wherein the porous device is a
membrane, preferably a polycarbonate membrane.
24. A method according to claim 1, wherein the porous device,
preferably the membrane is supported by at least one drain disk
from at least one side.
25. A method according to claim 24, wherein the at least one drain
disk is supported by at least one frit.
26. A method according to claim 1, wherein the nozzle of step (c)
is an orifice nozzle.
27. A method according to claim 1, wherein the nozzle of step (c)
has a diameter between about 0.05 mm and about 1 mm, preferably
between about 0.1 mm and about 0.2 mm.
28. A method according to claim 1, wherein the suspension or
solution is extruded through the porous device in step (b) and is
subsequently passed through the nozzle in step (c) with a flow rate
between about 1 ml/min and 1000 ml/min, preferably about 20 ml/min
and 100 ml/min.
29. A method according to claim 1, wherein the suspension or
solution is extruded through the porous device in step (b) and is
subsequently passed through the nozzle in step (c) with a pressure
between about 0.5 bar and 1200 bar, preferably about 5 bar and 600
bar.
30. A method according to claim 3, wherein the dehydration step (d)
is performed with a stream of gas, preferably heated gas.
31. A method according to claim 30, wherein the gas is an inert
gas, preferably nitrogen.
32. A method according to claim 3, wherein the dehydration step (d)
is performed by spray-drying, preferably in a pass-through or loop
operation.
33. A method according to claim 3, wherein the dehydration step (d)
is performed by a spray-freeze drying, whereby freezing is achieved
by contacting the liposomes obtained in step (c) with cryogenic
fluid.
34. A method according to claim 33, wherein the cryogenic fluid is
a cold gas or a cryogenic liquid, preferably nitrogen.
35. A method according to claim 33, wherein frozen particles
resulting from said spray freezing are subsequently dehydrated,
preferably under reduced pressure.
36. Liposomes prepared by the method of claim 1.
37. Dehydrated liposomes prepared by the method of claim 3,
comprising an average diameter smaller than about 500 nm,
preferably smaller than about 200 nm after rehydration in an
aqueous medium by mixing said dehydrated liposomes with said
aqueous medium.
38. Dehydrated liposomes according to claim 37, comprising a
polydispersity index of about lower than 0.2, preferably of about
lower than 0.15 after rehydration in an aqueous medium by mixing
said dehydrated liposomes with said aqueous medium.
39. Dehydrated liposomes according to claim 37, which are comprised
in dry particles with an average size between about 10 .mu.m and
about 1000 .mu.m, preferably between about 50 .mu.m and about 500
.mu.m.
40. A method for delivering an active agent to a subject comprising
administering the liposomes according to claim 36 to a subject.
41. An apparatus for the preparation of liposomes comprising a
porous device which is connected to a nozzle.
42. An apparatus according to claim 41 comprising (i) a container
for supplying a solution or suspension comprising lipids, (ii) a
porous device, (iii) a nozzle, (iv) means for connecting the
container (i) with the porous device (ii) by a conduit, (v) means
for generating pressure, and (vi) optionally means for dehydrating
the solution or suspension comprising liposomes and (vii)
optionally means for collecting the obtained liposomes.
43. An apparatus according to claim 41, wherein the nozzle is an
orifice nozzle.
44. An apparatus according to claim 41, wherein the nozzle has a
diameter between about 0.05 mm and about 1 mm, preferably between
about 0.1 mm and about 0.2 mm.
45. An apparatus according to claim 41, wherein the nozzle is
comprised in the means for dehydration.
46. An apparatus according to claim 41, wherein the porous device
has a pore size of between 50 nm and about 300 nm, preferably
between about 100 nm and about 200 nm.
47. An apparatus according to claim 41, wherein the porous device
is a membrane, preferably a polycarbonate membrane.
48. An apparatus according to claim 41, wherein said membrane is
supported by at least one drain disk from at least one side.
49. An apparatus according to claim 48, wherein the at least one
drain disk is supported by at least one frit.
Description
INTRODUCTION
[0001] The present invention relates to a method for the
preparation of liposomes in a single-pass mode. The method
comprises the extrusion of a solution or suspension comprising
lipids through a porous device an subsequently passing said
solution or suspension through a nozzle.
[0002] Liposomes are lipid vesicles of closed bilayers entrapping
an aqueous volume. Liposomes may be unilamellar vesicles,
constituted by a single bilayers membrane, or multilamellar
vesicles (MLV), constituted by multiple concentric bilayers.
Depending on their size, unilamellar vesicles are roughly
classified into small unilamellar liposomes (SUV) with an particle
size between about 25-100 nm and large unilamellar liposomes (LUV)
with an particle size between about 100 nm-10 .mu.m. For the
pharmaceutical use unilamellar liposomes are preferred over
multilamellar liposomes.
[0003] In recent years liposomes have become an important tool in
the pharmaceutical industry for the delivery of drugs. Hydrophobic
drugs are formulated in liposomes by the integration of the drug
into the lipid bilayers. Hydrophilic agent may be formulated in
liposomes by encapsulation in the aqueous core of the liposomes.
Liposomes are capable of influencing pharmacokinetics by a
sustained release of the drug to the body or reduce side effects by
limiting the free concentration of a drug. By attaching ligands to
the liposome or rendering their charge, liposomes facilitate a
targeted delivery of drugs to a desired site of action. Beside the
pharmaceutical use, liposomes are also frequently used for cosmetic
products.
[0004] For food applications liposomes have been used to deliver
food flavors and nutrients and more recently have been investigated
for their ability to incorporate food antimicrobials that could aid
in the protection of food products against growth of spoilage and
pathogenic microorganisms.
[0005] For the pharmaceutical administration of liposomal
preparations the particle size is a parameter of major concern. The
optimal size range for parenteral administration is between about
70 and 400 nm. Within this size range liposomes favour
biodistribution into certain target organs such as liver, spleen
and bone marrow. Furthermore liposomes within this size-range have
a good stability in blood and display predictable dug-release
rates. Liposomes of greater than 400 nm have a greater tendency to
aggregate. At a size of about 5 .mu.m, liposomes injected into the
circulation may block capillaries. Thus, size distribution is an
important topic in the application of liposomes.
[0006] A variety of principal procedures have been developed for
the initial dispersion of lipids in an aqueous system for the
preparation of liposomal suspensions. Commonly used methods include
the hydratisation of a dry lipid film with a desired aqueous medium
("film method", "hand shaken method") or the dispersion of an
organic solvent solution of lipids into an aqueous medium
("ethanol/ether-injection"). The resulting liposomal suspensions
usually have a broad size distribution and heterogeneous
lamellarity. Predominantly MLV can be observed as the initial
product. Different size processing methods have been developed to
produce liposomes of desired size range and uniformity.
[0007] Liposomes may be sized by sonication of larger and
multilamellar liposomes. Resulting liposomes are SUV in the size
range of about 25-80 nm. However, a narrow size distribution can
only be achieved at liposome sizes of about less than 50 nm. Since
SUV have only a limited drug loading capacity and unfavourable
pharmacokinetic and pharmakodynamic properties, sonication is not a
suitable method for liposomes destined for a pharmaceutical
applications. Other drawbacks of this method are the limited
processing volume, long sonication times and possible oxidation of
the lipids due to heat stress. Therefore, the method is not well
suitable for regular industrial manufacturing.
[0008] Alternatively, large multilamellar liposomes may be sized
and homogenized by a high pressure approach. In a discontinuous
process, a liposomal suspension is extruded at high pressure (up to
about 160 MPa) through a small orifice, for example using a "French
Press"--apparatus as described by Hunt and Papahadyopoulous (U.S.
Pat. No. 4,529,561) or by Brandi et al (WO 96/05808). This process
is repeated until the desired size distribution is achieved.
Homogenisation chambers or homogenisation nozzles have been
especially designed for the use with high pressure pumps as
disclosed for example in DE 3905354 or U.S. Pat. No. 6,331,314. The
"high pressure homogenisation" is also suitable for the preparation
of liposomes from lipid/water dispersions without the use of
organic solvent (Brandi, Martin M. et al, Liposome preparation
using high-pressure homogenizers. Liposome Technol. (2nd Ed.)
(1993), 1, 49-65). Typically SUVs with quite broad and variable
distribution are produced. The use of high pressure pumps has
enabled a cycling through an orifice at high pressure, rendering
possible the processing of larger batch sizes. Still, the process
is carried out in a discontinuous manner.
[0009] For the production of LUV with a defined size between about
100-400 nm and a narrow size distribution, methods of
size-processing based on the extrusion of liposomal suspension
through a uniform pore size membrane have been developed (Szoka et
al 1979, Olsen et al 1979). Preferably polycarbonate membranes,
usually of a pore size between about 50-200 nm are employed in this
method. The selection of the membrane pore size allows a
predetermination of the desired liposome size. To obtain liposomes
of high homogeneity, liposomes can be extruded through the membrane
multiple times (Cullis WO 86/00238, Goldbach P. et al.,
Spray-Drying of Liposomes for a Pulmonary Administration. II.
Retention of Encapsulated Materials (1993) Drug Development and
Industrial Pharmac, 19(9), 2623-2636). For the processing of larger
batch sizes, liposomal preparation can be extruded through the
membrane in a continuous recycling by a pump until the desired size
and polydispersity has been achieved (Janoff EP 0460720). In
general the polydispersity of the liposomes is deceased with
increasing numbers of extrusion cycles.
[0010] To obtain a narrow, well defined, size distribution of the
product and reduce the number of extrusion steps required, several
variations of the extrusion membrane are known in the prior art. By
applying a polymer membrane having a web-like structure for the
extrusion process, Morano et al U.S. Pat. No. 4,927,637 achieves
good polydispersity. Martin et al U.S. Pat. No. 4,737,232 discloses
an asymmetric membrane, whereas Suddith (U.S. Pat. No. 5,556,580)
uses a frit instead of a membrane for liposomal extrusion.
[0011] Different approaches to optimise the method employ high
pressure for the extrusion process. For example Sachse et al (DE
4328331) use pressures of 6.5 MPa for the extrusion through a
series of staggered membranes. Hill at (US 2005/0260256) enables
the use of up to about 55 MPa for the extrusion by using
hydrophilic screen membranes in a support cassette holder.
[0012] For the large scale production of liposomes with a suitable
size and narrow size distribution which has to be met for the
pharmaceutical application of drug loaded liposomes, usually
repeated processing of the liposomal material either by the high
pressure homogenisation or the membrane extrusion method is still
required. Hence, a single pass method for the continuous
preparation of appropriately sized liposomes can not be performed
with these standard methods.
[0013] Aqueous liposomal preparations are usually not stable over a
long period of time which is required for the industrial
manufacture and supply process. A standard method for preserving
liposomes is the dehydration of the aqueous liposomal preparations.
Dehydration may be achieved by spray-drying or spray-freeze-drying
which both requires the atomisation of the suspension.
[0014] It was the underlying problem of the current invention to
provide a continuous method for the preparation of liposomal
suspensions with a suitable size and size distribution for a
parenteral application. It was another problem to provide a
continuous method for the preparation of such liposomal suspensions
and the atomisation of the suspensions for the subsequent drying
procedure. Furthermore, scalability of the process and the use of
established standard material was desired.
SUMMARY OF THE INVENTION
[0015] The current invention provides a continuous method for the
preparation of liposomes from a suspension or solution. The
starting suspension or solution may comprise lipids which may
preferably be in the form of lipid particles and preferably also
comprising an aqueous medium. The lipid particles in the starting
suspension may be heterogeneously sized, whereas the liposomes
obtained by the method may be characterized by a homogeneous size.
The method comprises extruding the suspension or solution first
through a porous device and subsequently passing the suspension or
solution through a nozzle in a continuous mode. The method is
performed in a single-pass mode, wherein the suspension or solution
is extruded through the porous device and through the nozzle only
once. Preferably the liposomes obtained by the method comprise are
homogeneously sized. By the inventive process, the average particle
size distribution of lipid particles of the starting suspension is
reduced to a smaller particle size. The method may also include the
provision of the solution or suspension comprising lipids and the
collection of the prepared liposomes.
[0016] Thus, the invention relates to method for the preparation of
liposomes in a single-pass mode, comprising the steps: [0017] (a)
providing a suspension or solution comprising lipids, [0018] (b)
extruding the suspension or solution of step (a) through a porous
device, and subsequently [0019] (c) passing said suspension or
solution of step (b) through a nozzle, and [0020] (d) optionally
collecting the liposomes formed after step (c).
[0021] The method may be performed at high pressure of up to about
1200 bar, which is generated by suitable pumps. By restricting the
flow of the suspension or solution by means of a suitable nozzle,
the pressure of the suspension or solution is about equal on both
sides of the porous device. Preferably the porous device, through
which the liposomes are extruded, has a pore size of 50-300 nm,
more preferably of 100-200 nm.
[0022] Although homogenisation of liposomes by extrusion through
membranes or high pressure extrusion through nozzles or orifices
was known before, it could not be predicted, that the combination
of the two methods provides a continuous process suitable for a the
preparation of liposomes with a narrow size distribution and an
average size between 50 nm and 500 nm, preferably from about 50 nm
to about 200 nm. The latter product characteristics are preferred
for the use of liposomes in a parenteral medical application. To
obtain such characteristics by established extrusion techniques,
several extrusion steps have to be performed which prevents a
continuous set up of the process.
[0023] The extrusion of liposomes through a porous device with a
predetermined pore size is known as a restrictive method for the
sizing of liposomes. Therefore it was even more unexpected that the
size distribution of liposomes can be improved by subsequently
passing the liposomes through a nozzle. The size distribution of
the liposomes is characterized by the polydispersity index. Thus, a
decrease of the polydispersity index indicates a decrease of the
breadth of the size distribution.
[0024] Given that the suspension or solution comprising lipids is
maintained under high pressure after the porous device, passing
said suspension or solution through the nozzle enables a
spraying/atomisation. The atomisation may be performed in a device
that is suitable for either drying or freezing the atomised
suspension in a fashion commonly performed in a spray-drying or
spray-freezing process. Preferably, the drying/freezing may be
carried out by percolative drying as disclosed in co-owned European
application no. 06 022 538.0 "Percolative drug for the preparation
of particles" filed on 27 Oct. 2006, the content of which is herein
incorporated by reference.
[0025] It is a further aspect of the current invention to provide a
continuous method for the extrusion and spray-drying or
spray-freezing of liposomes, comprising the extrusion of a starting
suspension or solution comprising lipids through a porous device
and subsequently passing the suspension or solution through a
nozzle, thereby atomising the suspension or solution.
[0026] The invention furthermore discloses an apparatus comprising
a porous device, which is connected to a nozzle via a conduit. The
apparatus is suitable for performing the method as disclosed by the
invention.
DEFINITIONS
[0027] "About" in the context of amount values refers to an average
deviation of maximum +/-20%, preferably +/-10% based on the
indicated value. For example, an amount of about 30 mol % cationic
lipid refers to 30 mol %+/-6 mol % and preferably 30 mol %+/-3 mol
% cationic lipid with respect to the total lipid/amphiphile
molarity.
[0028] "Active compound" refers to a compound, or mixture of
compounds, which has a particular bioactivity based on which it is
useful as an agent useful for the diagnosis, prevention, or
treatment of a human or animal disease or condition. Drug
substances, diagnostic compounds and vaccines are important
examples of active compounds according to the present
invention.
[0029] "Aqueous medium" or "aqueous liquid" is a liquid material
which contains water. The material may represent a single liquid
phase, or a two- or multiphase system, wherein the continuous phase
is liquid and contains water. An aqueous suspension, a dispersion
or an emulsion, wherein the continuous phase is aqueous are also
examples of aqueous medium. An aqueous medium which contains a
colloidal material is hereinafter sometimes referred to as an
aqueous colloidal dispersion or solution.
[0030] "Cryogenic fluids" are materials that are liquid in the
temperature range which is necessary to freeze aqueous solutions,
preferably they are applied at temperatures below -90.degree. C.
The cryogenic fluid can freeze particles and/or pellets in-situ by
getting in contact with the cryogenic fluid.
[0031] "Drain disk" is a device with a filter like structure with a
pore size, which is larger than the pore size of the adjacent
porous device.
[0032] "Dry" or "dehydrate" are used interchangeable and relate to
the process of removing an aqueous medium by evaporation or
sublimation.
[0033] "Formation of droplets" refers to the conversion of a
material into fine particles or pellets by measures like pressure
atomisation, two fluid atomisation, centrifugal droplets formation
and nozzles like e.g. piezo droplet forming devices, sound
activated-, magnetic- and electrostatic droplet forming
devices.
[0034] "Hydrophilic excipient" refers to a pharmaceutically
acceptable, pharmacologically substantially inert material with
hydrophilic properties. Hydrophilicity means that the hydrophilic
excipient should be substantially water soluble.
[0035] "Homogeneity" or "homogeneous" as used herein refers to a
narrow size distribution of a particle population. The high size
homogeneity or narrow size distribution is characterized by a low
polydispersity index.
[0036] "Lipid" refers to its conventional sense as a generic term
encompassing fats, lipids, alcohol-ethersoluble constituents of
protoplasm, which are insoluble in water. Lipids are composed of
fats, fatty oils, essential oils, waxes, steroid, sterols,
phospholipids, glycolipids, sulpholipids, aminolipids,
chromolipids, and fatty acids. The term encompasses both naturally
occurring and synthetic lipids. Preferred lipids in connection with
the present invention are: steroids and sterol, particularly
cholesterol, phospholipids, including phosphatidyl and
phosphatidylcholines and phosphatidylethanolamines, and
sphingomyelins. Where there are fatty acids, they could be about
12-24 carbon chains in length, containing up to 6 double bonds. The
fatty acids are linked to the backbone, which may be derived from
glycerol. The fatty acids within one lipid can be different
(asymmetric), or there may be only 1 fatty acid chain present,
e.g., lysolecithins. Mixed formulations are also possible,
particularly when the non-cationic lipids are derived from natural
sources, such as lecithins (phosphatidylcholines) purified from egg
yolk, bovine heart, brain, or liver, or soybean.
[0037] "Liposomes" are artificial lipid bilayer vesicles of various
sizes and structures. Unilamellar vesicles are liposomes defined by
a single lipid bilayer enclosing an aqueous space. In contrast,
oligo- or multilamellar vesicles comprise several membranes.
Typically, the membranes are roughly 4 nm thick and are composed of
amphiphilic lipids, such as phospholipids of natural or synthetic
origin. Optionally, the membrane properties can be modified by the
incorporation of other lipids such as sterols or cholic acid
derivatives. Liposomes with particularly flexible membranes based
on phospholipids with a low phase transition temperature (i.e.
below body temperature) are sometimes referred to as
transfersomes.
[0038] Depending on their diameter and number of bilayer membranes,
liposomes may also be classified as multilamellar vesicles (MLV,
two or more bilayers, typically above approx. 150 to 200 nm), small
unilamellar vesicles (SUV, one single bilayer, typically below
about 100 nm), multivesicular vesicles (MVV, several vesicular
structures within a larger vesicle), and large unilamellar vesicles
(LUV, one single bilayer, typically larger than about 100 nm).
[0039] For cosmetic application of liposomal the particle size is a
parameter of minor concern. The size range for dermal
administration is between about 70 and 4000 nm. Within this size
range liposomes favour application of dermal and transdermal
use.
[0040] A "nozzle" as used herein refers to a conduit with a
variable cross-sectional area in which a fluid accelerates into a
high-velocity stream. The fluid must be compressed to a state of
high pressure before it is sent through the nozzle, where the
droplet break-up may result in atomization.
[0041] "Paclitaxel" (which should be understood herein to include
analogues, formulations, and derivatives such as, for example,
deacetylpaclitaxel, deacetyl-7-epipaclitaxel, docetaxel, taxotere
(a formulation of docetaxel), 10-desacetyl analogs of paclitaxel
and 3'N-desbenzoyl-3'N-t-butoxycarbonyl analogs of paclitaxel) may
be readily prepared utilizing techniques known to those skilled in
the art (see also WO 94/07882, WO 94/07881, WO 94/07880, WO
94/07876, WO 93/23555, WO 93/10076; U.S. Pat. Nos. 5,294,637;
5,283,253; 5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529;
and EP 590,267), or obtained from a variety of commercial sources,
including for example, Sigma Chemical Co., St. Louis, Mo. (T7402
from Taxus brevifolia; or T-1912 from Taxus yannanensis).
Paclitaxel should be understood to refer to not only the common
chemically available form of paclitaxel, but analogs (e.g.,
taxotere, as noted above) and paclitaxel conjugates (e.g.,
paclitaxel-PEG, paclitaxeldextran, or paclitaxel-xylose).
[0042] The term "taxane" as used herein refers to the class of
antineoplastic agents having a mechanism of microtubule action and
having a structure that includes the unusual taxane ring structure
and a stereospecific side chain that is required for cytostatic
activity. Also included within the term "taxane" are a variety of
known derivatives, including both hydrophilic derivatives, and
hydrophobic derivatives. Taxane derivatives include, but not
limited to, galactose and mannose derivatives described in
International Patent Application No. WO99/18113; piperazino and
other derivatives described in WO 99/14209; taxane derivatives
described in WO99/09021, WO 98/22451, and U.S. Pat. No. 5,869,680;
6-thio derivatives described in WO 98/28288; sulfenamide
derivatives described in U.S. Pat. No. 5,821,263; and taxol
derivative described in U.S. Pat. No. 5,415,869.
[0043] As used herein, "thermally sensitive",
"temperature-sensitive" or "thermally labile" means that a material
is incompatible with drying methods based on the evaporation of
water by heat, such as spray drying. In other words, a thermally
labile material looses at least some of its structure, activity,
functionality or chemical purity when treated by such thermal
drying methods, so that the product may be pharmaceutically
unacceptable.
[0044] "Pharmaceutically acceptable" is meant to encompass any
substance, which does not interfere with effectiveness of the
biological activity of the active ingredient and that is not toxic
to the host to which it is administered.
[0045] "Polydispersity" is the width of the distribution of the
particle size in a given particle sample. The Polydispersity is
characterised by the polydispersity index.
[0046] "Polydispersity index" (PDI) is an estimation of the wide of
the distribution of the particle size in a given liposomal sample.
Before calculation of the PDI the particle size will be determined
by dynamic light scattering (DLS). This technique measures the
time-dependent fluctuations in the intensity of scattered light
which occur because the particles are undergoing Brownian motion.
Analysis of these intensity fluctuations enables the determination
of the diffusion coefficients of the particles which are converted
into a size distribution. The calculations of these parameters are
defined in the ISO standard document 13321:1996 E.
[0047] "Single-pass mode" or "single-pass process" refers to a
method wherein the processed material passes a processing step only
one time. Thus, the suspension, solution or liposomes employed in
the inventive method pass the porous device or the nozzle employed
in the current invention only once.
[0048] Lipid particles used in the current invention may be
liposomes, lipid complexes, solid lipid nanoparticles, lipoplexes,
niosomes, micelles or mixed micelles, oligo- or multilamellar
vesicles. Preferably, the lipid particles are liposomes. It is the
most preferred embodiment of the invention that the liposomes are
cationic liposomes. The method might also employ particles formed
through the association of amphiphilic molecules such as
detergents. In contrast to liposomes, these structures are based on
monolayers which are less stable and tend to disassemble upon
dilution. Micelles are e.g. disclosed in WO 02/085337 and EP-A 730
860, the teachings of which are incorporated herein by
reference.
[0049] The lipids or lipid particles are provided in a suspension
or solution comprising an aqueous medium. Besides water, the
aqueous medium of the present invention may comprise one or more
further liquid constituents which are at least partially miscible
with water, preferably organic solvents, more preferably alcohols
(e.g. C.sub.1-4 alcohols such as methanol, ethanol, propanol,
butanol and combinations thereof, etc.) or ketones (e.g. C.sub.1-4
ketones such as acetone, methylethyl-ketone, DMF, DMSO and
combinations thereof, etc.). In a preferred embodiment of the
invention, the aqueous medium is water or a mixture of water and
ethanol. The ratio between water and the further liquid constituent
is preferably between about 99.9:0.1 and about 10:90 (v/v), more
preferably between about 70:30 and about 40:60 (v/v), most
preferably between about 80:20 and about 60:40 (v/v).
[0050] The use of said organic solvents, preferably ethanol in the
ratio described, in the combination with the further embodiments of
the invention provides a variety of advantages over previously
known systems for producing unilamellar liposomes, including the
following:
1) the method results in very small and homogenous liposomes; 2)
the ability to use high lipid concentrations (e.g., on the order of
100 mM) so as to easily achieve high loading efficiencies; 3) a
decreased viscosity, which leads to an increased fluidity, enabling
high flow rates of the lipid suspension or solution; 4) the use of
organic solvents leads to a reduction in drying temperature during
the evaporation process.
[0051] The liposomes prepared by the inventive method may have
different sizes, lamellarity and structure. Preferably the
liposomes prepared by the method of the invention have an average
diameter of about 50 nm to about 500 nm. Most preferred is a size
of about 50 to about 200 nm. Preferably the liposomes are
unilamellar liposomes.
[0052] The suspension or solution, lipid particles or liposomes
used within the context of the present invention may comprise
neutral, anionic and/or preferably cationic lipids. Cationic lipids
are preferably comprised in an amount of at least about 30 mol %,
more preferably of at least 40 mol % and most preferably in an
amount of at least 50 mol % of total liposome forming lipids.
Neutral or anionic lipids may be selected from sterols or lipids
such as cholesterol, phospholipids, lysolipids, lysophospholipids,
sphingolipids or pegylated lipids with a neutral or negative net
charge. Useful neutral and anionic lipids thereby include:
phosphatidylserine, phosphatidylglycerol, phosphatidylinositol (not
limited to a specific sugar), fatty acids, sterols, containing a
carboxylic acid group for example, cholesterol,
1,2-diacyl-sn-glycero-3-phosphoethanolamines, including, but not
limited to, 1,2-dioleylphosphoethanolamine (DOPE),
1,2-dihexadecylphosphoethanolamine (DHPE),
1,2-diacyl-glycero-3-phosphocholines, including, but not limited to
1,2-distearylphosphatidylcholine (DSPC),
1,2-dipalmitylphosphatidylcholine (DPPC),
1,2-dimyristylphosphosphatidylcholine (DMPC), phosphatidylcholine
preferably egg PC, soy PC and sphingomyelin. The fatty acids linked
to the glycerol backbone are not limited to a specific length or
number of double bonds. Phospholipids may also have two different
fatty acids. Preferably the further lipids are in the liquid
crystalline state at room temperature and they are miscible (i.e. a
uniform phase can be formed and no phase separation or domain
formation occurs) with the used cationic lipid, in the ratio as
they are applied. In a preferred embodiment the neutral lipid is
1,2-dioleylphosphatidylcholine (DOPC). Further, the neutral and/or
anionic lipids may comprise poly(ethylene)glycol chains.
[0053] Cationic lipids may preferably be selected from a group
comprising N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium
(TAP) salts, preferably the chloride or methylsulfate. Preferred
representatives of the family of TAP lipids are DOTAP (dioleoyl-),
DMTAP (dimyristoyl-), DPTAP (dipalmitoyl-), or DSTAP (distearoyl-).
Other useful lipids for the present invention may include, but are
not limited to: DDAB, dimethyldioctadecyl ammonium bromide;
1,2-diacyloxy-3-trimethylammonium propanes, (including but not
limited to: dioleoyl, dimyristoyl, dilauroyl, dipalmitoyl and
distearoyl; also two different acyl chains can be linked to the
glycerol backbone); N-[1-(2,3-dioloyloxy)propyl]-N,N-dimethyl amine
(DODAP); 1,2-diacyloxy-3-dimethylammonium propanes, (including but
not limited to: dioleoyl, dimyristoyl, dilauroyl, dipalmitoyl and
distearoyl; also two different acyl chain can be linked to the
glycerol backbone);
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
(DOTMA); 1,2-dialkyloxy-3-dimethylammonium propanes, (including but
not limited to: dioleyl, dimyristyl, dilauryl, dipalmityl and
distearyl; also two different alkyl chain can be linked to the
glycerol backbone); dioctadecylamidoglycylspermine (DOGS);
3.beta.-[N--(N',N'-dimethylamino-ethane)carbamoyl]cholesterol
(DC-Chol);
2,3-dioleoyloxy-N-(2-(sperminecarboxamido)-ethyl)-N,N-dimethyl-1-propanam-
inium trifluoro-acetate (DOSPA); .beta.-alanyl cholesterol; cetyl
trimethyl ammonium bromide (CTAB); diC14-amidine;
N-tert-butyl-N'-tetradecyl-3-tetradecylamino-propionamidine;
14Dea2; N-(alpha-trimethylammonioacetyl)didodecyl-D-glutamate
chloride (TMAG);
O,O'-ditetradecanoyl-N-(trimethylammonio-acetyl)diethanolamine
chloride; 1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide
(DOSPER);
N,N,N',N'-tetramethyl-N,N'-bis(2-hydroxylethyl)-2,3-dioleoyloxy-1,4-butan-
e-diammonium iodide;
1-[2-(acyloxy)ethyl]2-alkyl(alkenyl)-3-(2-hydroxyethyl)-imidazolinium
chloride derivatives as described by Solodin et al. (Solodin et
al., 1995), such as
1-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)-heptadecenyl-3-(2-hydroxyethyl)-
imidazolinium chloride (DOTIM),
1-[2-(hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imidazolinium
chloride (DPTIM), 2,3-dialkyloxypropyl quaternary ammonium compound
derivatives, containing a hydroxyalkyl moiety on the quaternary
amine, as described e.g. by Feigner et al. (Feigner et al., 1994)
such as: 1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide
(DORI), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium
bromide (DORIE), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypropyl
ammonium bromide (DORIE-HP),
1,2-dioleyl-oxy-propyl-3-dimethyl-hydroxybutyl ammonium bromide
(DORIE-HB), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypentyl ammonium
bromide (DORIE-Hpe),
1,2-dimyristyloxypropyl-3-dimethyl-hydroxylethyl ammonium bromide
(DMRIE), 1,2-dipalmityloxypropyl-3-dimethyl-hydroxyethyl ammonium
bromide (DPRIE), 1,2-disteryloxypropyl-3-dimethyl-hydroxyethyl
ammonium bromide (DSRIE); cationic esters of acyl carnitines as
reported by Santaniello et al. (U.S. Pat. No. 5,498,633); cationic
triesters of phospahtidylcholine, i.e.
1,2-diacyl-sn-glycerol-3-ethylphosphocholines, where the
hydrocarbon chains can be saturated or unsaturated and branched or
non-branched with a chain length from C.sub.12 to C.sub.24.
Preferably, the cationic lipids comprise two acyl chains, which may
be different or identical.
[0054] It is an aspect of the present invention that the starting
suspension or solution comprising lipids, preferably lipid
particles, and the liposomes obtained by the inventive method may
comprise an active compound. Preferably, the active compound is a
pharmaceutically active compound. The active compound may be
incorporated within or associated with the liposomes and/or lipid
particles. In the case of liposomes, for example, a hydrophilic
active compound may be encapsulated within the aqueous inner space
of the liposomes, whereas a lipophilic active compound is usually
associated with the membrane-forming lipids. In the case of lipid
nanoparticles, lipophilic compounds are also easily incorporated
within the matrix of such nanoparticles.
[0055] Liposomes and/or lipid particles are particularly suitable
drug carriers for active compounds, in particular pharmaceutically
active compounds, which are not easy to deliver effectively, such
as very poorly soluble compounds, sensitive (including thermally
labile) active compounds, peptides, proteins, and nucleic acids
including DNA, RNA, iRNA, siRNA, or oligonucleotides. Another
preferred group of active compounds are those which are more
effectively delivered in colloidal form because the distribution of
colloids in the organism after intravenous injection is more
favorable with regard to efficacy or side effects than the
administration of a solution of the compound. Preferred examples of
pharmaceutically active compounds include, but are not limited to,
antimicrobial, antifungal, antiviral, and cytostatic or cytotoxic
agents. Examples of such agents include doxorubicin, mitoxanthrone,
and amphotericin B.
[0056] Liposomes used within the context of the invention are
especially suitable for the encapsulation of hydrophobic drugs.
Examples are taxanes, camptothecins, doxorubicin, michellamine B,
vincristine, and platinum compounds such as cisplatin. Preferred
examples of such hydrophobic drugs are paclitaxel, docetaxel and
camptothecins, which have a low solubility in water and/or are heat
sensitive.
[0057] In a specially preferred embodiment of the invention, the
liposomes obtained by the method comprise DOTAP used in the form of
DOTAP chloride, DOPC and paclitaxel, preferably in a ratio of about
50:47:3. This formulation is also designated MBT-0206 or EndoTAG-1.
EndoTAG-1 has a lipid content of about 10 mM in a about 10% m/m
trehalose dihydrate solution. The preparation of such a liposomal
preparation is disclosed in WO 2004/002468.
[0058] The liposomes obtained according to the present invention
can be administered orally, transdermally, intravenously,
intrabronchially, intramuscularly, intraoculary, subcutaneously and
interperitoneally. As a drug delivery system, liposomes can
significantly change the pharmacokinetic and pharmacodynamic fate
of a compound by enhancing drug uptake, delaying the loss of
rapidly cleared drugs and reducing drug toxicity. They have widely
been investigated for the delivery of chemotherapeutic agents for
treatment of cancer, antimicrobial agents for treatments of
bacterial, viral and parasitic diseases, and also for use as
immunological adjuvans for delivery of vaccines.
[0059] The active compound comprised in the liposomes may also be a
compound which is effective for a cosmetic purpose. Such compounds
may be especially compounds that have an effect on the human skin
or hair. The compound which has a cosmetic purpose may also be one
or more lipids constituting the liposome.
[0060] Liposomes may be labile in various respects. Their physical
structure may be sensitive to heat, so that a highly thermal drying
method would lead to the disassembly of the liposomal membranes, to
the loss of functionality or to the loss of incorporated or
associated active compound. Depending on their composition, they
may also be chemically labile to heat. For example, some lipids
hydrolyse or oxidise rapidly in an aqueous environment at elevated
temperatures.
[0061] In a preferred embodiment, the starting suspension or
solution comprising lipids or lipid particles optionally comprises
a hydrophilic excipient. Useful hydrophilic excipients can be
monomeric, oligomeric or polymeric and may be found among several
chemical classes of compounds. According to one of the preferred
embodiments of the invention, the hydrophilic excipient is a
saccharide, e.g. a mono-, di-, oligo- or polysaccharides, a sugar
alcohol, an amino acid, a peptide, a protein, a water-soluble
polymer or a combination thereof.
[0062] A saccharide, or carbohydrate, is defined as a compound
predominantly composed of carbon, hydrogen, and oxygen. Useful
saccharides include sugar and sugar alcohols, oligosaccharides,
water soluble polysaccharides and derivatives thereof. Preferred
saccharides according to the invention include, but are not limited
to, glucose, fructose, lactose, sucrose, trehalose, maltose,
cellobiose, galactose, maltotriose, maltopentose, raffinose,
dextrin, dextran, inulin, mannitol, sorbitol, xylitol, chitosan;
water soluble cellulose derivatives such as methylcellulose,
hydroxypropylcellulose, hydroxyethylcellulose, and hypromellose;
alginates, soluble starches or starch fractions, xanthan gum, guar
gum, pectin, carrageen, galactomannan, gellan gum, tragacanth,
including any derivatives of these. Particularly preferred
saccharides are glucose and trehalose.
[0063] Other useful hydrophilic excipients may be selected from
other chemical classes, such as from water soluble amino acids,
peptides or proteins. For example, glycine or other natural amino
acids may be used. Useful proteins include, but are not limited to,
gelatine, albumin, whey protein, soy protein, or other food or
vegetable proteins.
[0064] Still further examples of useful hydrophilic excipients are
polymers such as water soluble polymers such as solid polyethylene
glycols, polyvinylalcohol, polyacrylates, or
polyvinylpyrrolidone.
[0065] According to the invention, mixtures of more than one
hydrophilic excipient may be used. For example, there may be a need
to adjust several parameters such as pH, solubility, and
wettability independently. In this case, a first hydrophilic
excipient may be selected as a basic carrier material for the
colloidal systems, whereas one or more further hydrophilic
excipients may be incorporated to obtain a certain pH and/or
wettability.
[0066] The content of the hydrophilic excipient, or mixture of
hydrophilic excipients, in the aqueous phase may vary widely,
depending on its aqueous solubility and other considerations, and
be even up to about 80 wt.-%. More preferably, it is from about 0.1
to about 65 wt.-%. A content of about 3 to about 60 wt.-%, and
particularly from about 5 to about 50 wt.-% is preferred.
[0067] Of course, the aqueous medium may comprise further
excipients or auxiliary substances, which may or may not be
hydrophilic or water soluble. Depending on their nature and that of
the extraction medium, i.e. on whether or not these substances are
soluble in and extracted by the extraction medium, such substances
can be comprised into the dry particles or removed together with
the water and organic solvent. Substances, which are comprised into
the dry particles, should be pharmaceutically acceptable.
[0068] Further excipients which may be useful in particles
formulations, and in particular in particles formulations which are
used for reconstitution and optionally, parenteral administration,
are generally known to pharmaceutical formulation scientists.
Preferred further excipients include stabilisers, surfactants,
wetting agents, bulking agents, lyophilisation aids, antioxidants,
chelating agents, preservatives, osmotic agents, acidic or alkaline
excipients for adjusting the pH, etc.
[0069] Among the preferred excipients according to the invention
are stabilisers and antioxidants. Antioxidants may prevent the
oxidation of an incorporated active compound, but also that of
components of the colloidal in particular if lipids are used which
are sensitive to oxidisation. Useful compounds include, for
example, lipid-soluble antioxidants such as alpha-, beta-, and
gamma-tocopherol, ubiquinol, lycopene, alpha- and beta-carotene,
nordihydroguaiaretic acid, butyl hydroxyanisole, butyl
hydroxytoluene, ethylenediamine tetraacetic acid,
diethylenetriamine pentaacetic acid, desferal, p-hydroxybenzoic,
vanillic, syringic, 3,4-dihydroxybenzoic, p-coumaric, ferulic,
sinapic and caffeic acids, ascorbyl palmitate, carnosol, esculetin,
esculin, fraxetin, fraxin, quercetin, morin, etc. Particularly
preferred are alpha-tocopherol and ethylenediamine tetraacetic
acid, including the pharmaceutically acceptable derivatives
thereof. On the other hand, if chemically pure, semisynthetic or
synthetic saturated lipids are used for composing the colloidal
systems, no antioxidant may be needed.
[0070] As described before, it is an aspect of the current
invention, that a starting suspension or solution comprising lipids
and/or lipid particles is extruded through a porous device and
subsequently passed through a nozzle in a continuous mode. The
utilization of a nozzle with a sufficiently small diameter
restricts the flow of the suspension after being extruded through
the porous device and thereby pressurizes the suspension. The
sequential use of a porous device and a nozzle enables an about
equally high pressure on both sides of the porous device when the
inventive method is performed.
[0071] It is also an aspect of the invention to disclose an
apparatus which is suitable to carry out the inventive method. The
apparatus comprises at least one porous device which is connected
to at least one nozzle and at least one pump, suitable for passing
an suspension or solution comprising lipids through the porous
device and the nozzle.
[0072] Thus, it is a further aspect of the invention to provide an
apparatus for the preparation of liposomes comprising: [0073] (i) a
container for supplying a solution or suspension comprising lipids,
[0074] (ii) a porous device, [0075] (iii) a nozzle, [0076] (iv)
means for connecting the container (i) with the porous device (ii)
by a conduit, [0077] (v) means for generating pressure, and [0078]
(vi) optionally means for dehydrating the solution or suspension
comprising liposomes and [0079] (vii) optionally means for
collecting the obtained liposomes.
[0080] An illustration of preferred embodiments of the apparatus
according to the present invention are shown in FIGS. 1 and 2.
[0081] The porous device used to prepare the liposomes by the
inventive method or which is part of the inventive apparatus
preferably has a pore size of between about 300 nm and about 50 nm,
more preferably between about 200 nm and about 100 nm. The porous
device comprises at least one porous layer, i.e. the porous device
may comprise one porous layer or a plurality of identical or
different porous layers. The porous device may be a membrane, a
filter, a frit or any other restricting device comprising pores and
combinations thereof. The device may consist of a polymeric
material, metal, glass, ceramic or any other suitable material. The
material may be chemically inert, low absorbing for the
constituents of the solution/suspension and wet able. In a
preferred embodiment, the porous device is a membrane, preferably a
polycarbonate membrane. In another embodiment the porous device may
be a sterile filtration membrane. Polycarbonate membranes produced
by Osmonics Inc., USA have been found to work successfully in the
practice of the present invention. According to the present
invention, the pressure on both sides of the porous device is
preferably substantially identical.
[0082] Because the suspension is passed trough the porous device
system only once, the porous device does not have to be changed due
to pore clogging.
[0083] Clogging in general depends on such variables as lipid
composition, purity and concentration, as well as on the pressure
and flow rates used. A second passage through a porous device is in
no case objective of this invention.
[0084] The porous device, preferably a membrane, that is used for
extrusion, may be supported by at least one drain disk on at least
one side, preferably on both sides embedding the porous device.
Drain disks of this type produced by Osmonics Inc., USA have been
found to work successfully in the practice in the present
invention.
[0085] The at least one drain disk may be further mechanically
supported by at least one frit, especially on the porous device's
side which is connected to the nozzle. Such types of membrane
holders are generally known to the skilled worker in the art.
[0086] The frit element is a structural member comprised of
generally bead-like material partially conjoined, usually by
compression and/or sintering. Among the many materials useful in
frit preparation are ceramic, glass metal and metal/carbide
bead-like materials which are conjoined into a frit. Due to the low
affinity of lipid for metal, metal frits are preferred for
facilitating substantially total through-put of material. The
preferred metal frit is of stainless steel. Paths through the frit
are not straight paths and path walls are irregular. Frits may be
prepared in a number of shapes including rod or disc shapes.
Suitable frits are available from a number of sources such as Ace
Scientific Company, (East Brunswick, N.J.), Scientific Systems,
Inc. (State College, Pa.) and Ranin Instrument Company (Woburn,
Mass.). Frits are characterized in having non-uniform pore size
over a pore size range. Frit pore size range may be experimentally
determined on the basis of material that passes through or is
excluded by the frit. This defines a range of particle sizes a
portion of which lie above and below, the stated pore size.
Preferred pore sizes are .gtoreq.1 .mu.m, pref. 1-50 .mu.m, more
preferably 5-20 .mu.m.
[0087] The increased strength of the embedded porous device,
preferably of the embedded membrane which is supported by drain
disks and optionally at least one frit, enables high flow rate of
extrusion through the membrane. The porous device, preferably the
membrane does not suffer occlusion and is not deformed by the
pressure of the extrusion step. Surprisingly membrane type
extrusion is not rate limited by the minimal resistance to
deformation of membrane material.
[0088] A very preferred embodiment of the present invention is
shown in FIG. 2, wherein the assembly of a preferred porous device
is illustrated. In particular, this preferred porous device
comprises a membrane, preferably a polycarbonate membrane (14)
which is supported on both sides by drain disks (13). Further, the
preferred porous chain comprises a further porous material,
preferably a frit, which is located on the porous device's side
which is connected with the nozzle.
[0089] It is a further aspect of the current invention, that after
the extrusion through the porous device, the suspension or solution
of lipids/liposomes is subsequently passed through a nozzle. Useful
nozzles to carry out this step of the method of the invention or
which are part of the inventive apparatus are also generally known
to the skilled worker in the field. They include, for example,
rotating disk nozzles, impinging jet nozzles, capillary nozzles,
single orifice nozzles, ultrasonic nozzles of vibrating or
pulsating type, two-fluid nozzles such as coaxial two-fluid nozzles
etc. In a preferred embodiment of the invention the nozzle is an
orifice nozzle. The nozzle used to further improve the liposomes or
the unimodal distribution of liposomes was an orifice nozzle 121 VG
1/4 produced by Schlick atomization technologies, Untersiemau,
Germany. Within the current invention, the preferred pore size of
the nozzle is between about 0.05 mm to about 1 mm, more preferably
between about 0.1 mm to about 0.2 mm, but also nozzles with a
greater or smaller pore size might be used. In the inventive
apparatus, the nozzle may be comprised in a container suitable for
dehydrating the obtained liposome, in particular suitable for
dehydration by spray-drying or spray-freeze drying.
[0090] The flow rate of the solution or suspension may be in the
range of about 1 ml/min to about 1000 ml/min. More typically, the
liposomes were prepared by the method of the invention applying a
flow rate of 10 ml/min to 200 ml/min, and more preferably from
about 20 ml/min to about 100 ml/min.
[0091] According to another embodiment, the pressure used for
extruding the suspension or solution comprising lipids through the
porous device and the pressure for passing the suspension or
solution through the nozzle may be substantially identical, in
particular may be between 0.5 bar and 1200 bar (5.times.10.sup.4 Pa
and 1.2.times.10.sup.8 Pa). More typically, the liposomes can be
prepared by the method of the invention with 5 bar to 600 bar
(5.times.10.sup.5 Pa to 6.times.10.sup.7 Pa, preferably from about
10 bar to about 500 bar (1.times.10.sup.6 Pa to about
5.times.10.sup.7 Pa and more preferably from about 20 bar to about
150 bar (2.times.10.sup.6 Pa to about 1.5.times.10.sup.7 Pa).
[0092] Pumping the lipid/liposome suspension or solution with the
pressure and flow rate employed in the inventive method as
described above may be achieved by suitable high pressure pumps
that are generally known in the art. When piston pumps are
employed, the suspension may be drawn into the pump head itself.
External pumping of feed stock from the feed reservoir by an
external pumping device is possible. The invention is not limited
to piston pumps and any suitable pumping means may be used
including diaphragm pumps. In embodiments with a sufficient
hydrostatic head and thus a sufficient flow rate and pressure
differential the pump is not necessary.
[0093] It is another aspect of the current invention that the use
of pressures between about 10 bar and about 500 bar
(1.times.10.sup.6 Pa and about 5.times.10.sup.7 Pa) enables the
preparation of liposomes using a starting suspension or solution
comprising a high lipid concentration of up to about 400 mM lipid.
By using high pressure for the porous device extrusion, clogging of
the extrusion porous device can be prevented even at high lipid
concentrations. Prior art liposome sizing techniques employing
polycarbonate porous devices used pressures less than 7 bar, and
thus were limited to lipid concentrations of 60 .mu.mol/ml. In the
current process, rapid extrusion rates on the order of 20 ml/min
and above are still achieved for high lipid concentrations when
pressures are higher, e.g. when pressures in the range of 20 bar to
150 bar (2.times.10.sup.6 Pa and 1.5.times.10.sup.7 Pa) are
used.
[0094] The current method as described in its embodiments enables
the rapid preparation of liposomes in a continuous, reproducible
mode by a single extrusion through a porous device and subsequent
passing through a nozzle. The increase of the pressure used for
performing the method results in a reduced size and a narrower size
distribution of the liposomes prepared by the process.
[0095] It is another aspect of the current invention, that the
liposomes prepared by the inventive method have a high size
homogeneity as embodied by a narrow size distribution. The high
size homogeneity or narrow size distribution is characterized by a
low polydispersity index. Preferably the liposomes prepared by the
current invention are characterized by a polydispersity index lower
than about 0.3, more preferably lower than about 0.2, most
preferably lower than about 0.1. Polydispersity (PI) of the
liposomes may be determined by photon correlation spectroscopy
(PCS) using a Malvern Zetasizer Nano (Malvern Instruments; UK).
[0096] In another aspect of the invention, liposomes may be
dehydrated subsequent to their preparation to preserve the
liposomes for prolonged storage. Suitable methods for dehydrating
liposomes include spray-drying or spray-freeze-drying. Formation of
droplets by the atomisation of the liposome comprising suspension
represents the initial step in both methods of dehydration.
Liposomes my also be dehydrated under reduced pressure. In an
especially preferred embodiment, liposomes may be dehydrated by
percolative drying as described in the co-owned European patent
application no. 06 022 538.0 "Percolative drying for the
preparation of particles" filed on 27 Oct. 2006.
[0097] Its has been described above, that it is an object of the
present invention, that a suspension or solution comprising lipids,
preferably comprising lipid particles, most preferably liposomes is
passed through a nozzle, preferably at a high pressure. By this
way, the suspension is atomised, leading to a formation of droplets
from the suspension or solution.
[0098] Therefore, it is another aspect of the present invention, to
provide a continuous method for preparation of liposomes from a
starting suspension or solution comprising lipids, and dehydrating
said liposomes by spray-drying or spray-freeze-drying, whereby the
method comprises extruding a suspension or solution comprising
lipids through a porous device and subsequently passing the
suspension or solution through a nozzle, whereby the suspension is
atomised to form droplets.
[0099] Drying of the liposomes after droplet formation may be
achieved by contacting the droplets with a gas stream, preferably a
heated gas stream, to obtain solid particles. Preferably, the used
gaseous stream is an inert gas. The drying gas can preferably be a
low-oxygen gas containing less than 0.1 vol. %, preferably less
than 0.05 vol. %, oxygen or an oxygen-free gas. Inert gaseous are
increasing the safety of a heated drying systems that contains
highly flammable solutions, by pumping nitrogen, carbon dioxide,
helium, neon, argon, krypton, xenon and radon or some other inert
gas in order to displace oxygen. The effect of these systems is to
either completely remove the oxygen, or reduce it to a negligible
level. In a preferred embodiment of the invention nitrogen is used
as an inert gaseous. In an other embodiment of the invention the
inert gas protects the active ingredients and excipients containing
in the formulation. Preferably the spray-drying is performed in a
suitable device for spray drying. The spray-drying may for example
be performed in a drying tower. The dehydrated liposomes are
separated from the gas stream and collected.
[0100] The method according to the invention can be performed in a
pass-through or loop operation as known as inert-spray-drying
device to an a skilled person in the field. In the pass-through or
loop operation, inert gas can be heated as the drying gas. The
drying gas can then be used to spray dry the dryer feed of the
suspension containing the suspension with liposomes. The spray
drying can be performed under excess pressure, normal pressure or
partial vacuum. A favourable process pressure range can exist if
powder conforming to specification is produced with the drying gas
at the maximum permissible system temperature and at maximum
capacity. Since the introduction of oxygen into the system must be
avoided, the plant can preferably be operated under normal pressure
or slight overpressure of 0 to 200 mbar (2.times.10.sup.4 Pa) above
ambient pressure. The heated drying gas inside the process chamber
can display a temperature of 10.degree. C. to 500.degree. C. such
that the solvent evaporates when the dryer feed comes into contact
with the drying gas. More typically, the suspension were dried by
the method of the invention with 30.degree. C. to 250.degree. C.,
and more preferably from about 80.degree. C. to about 200.degree.
C.
[0101] Separation of the dried liposomal product can take place
with discharge of the product. The product stream can be separated
from the waste-gas stream by suitable means, for example using
filters or cyclones, optionally cooled and if necessary stored
under a protective gas atmosphere or packed. If surface filters
with pressure surge cleaning are used for dust collection, cleaning
can be performed with any oxygen-free gas, but preferably with
preheated inert gas or a split stream of the drying gas.
[0102] The solvents can be condensed and the exhaust air is
recycled by processing through the inert loop. In the pass-through
operation fresh drying gas is repeatedly fed into the process
chamber and the exhaust gas leaving the process chamber discarded.
The method according to the invention also can be performed in a
gas recycling operation in the loop operation the used drying gas
is free of solvents. The difference from the pass-through operation
is that the exhaust gas can be recycled and conditioned by energy
input in such a way that it can be used again as the drying gas.
During conditioning, the liquid components evaporated in the
process chamber can be partially removed from the exhaust gas such
that they too can be recycled. As complete as possible a gas
recycling with removal of the excess solvents and inert gases is
desirable from an economic perspective.
[0103] A preferred embodiment of an apparatus for the preparation
of dry liposomes according to the invention is shown in FIG. 1.
[0104] Freezing of the droplets in a spray-freezing step may be
achieved by contacting said droplets with a cryogenic fluid. The
cryogenic fluid can be a cold gas or a cryogenic liquid. Cryogenic
liquids are chilled liquids like argon, helium, hydrogen, nitrogen,
oxygen, methane, carbon dioxide, nitrous oxide, isopentane, hexane,
or ethanol and other fluids like hydrocarbon fluids or mixtures
thereof. In a preferred embodiment of the invention nitrogen is
used as a cryogenic liquid. The contacting of the droplets with the
cryogenic fluid results in the formation of frozen particles. Key,
variable operating parameters include rotor speed, atomising gas
pressure, annular gap gas pressure, air volume, solution spray-rate
and spray-gun nozzle diameter, slit width for the fluidised bed and
inlet and outlet air temperatures.
[0105] It is a feature of the present invention that the liposomes
comprised in the particles obtained by the freezing step may be
dehydrated under reduced pressure without thawing said particles.
Under reduced pressure, water and other solvents are removed from
frozen particles by sublimation. The reduction of pressure enables
decreased drying times for thermally labile compounds.
[0106] In order to obtain solid particles in which liposomes are
incorporated or embedded, it is useful to select a weight ratio of
the hydrophilic excipient to the colloidal systems which favours an
acceptable degree of incorporation of the colloid. For example, if
the aqueous liquid comprises more colloidal systems than
hydrophilic excipient, some of the colloidal systems may not be
incorporated in or embedded in the solid particles produced by the
method of the invention. Therefore, it is preferred that the weight
ratio of the hydrophilic excipient to the colloidal system is at
least about 1, and more preferably at least about 2, such as about
3 to about 5 or even more. On the other hand, an extremely high
ratio should be avoided because it might lead to rather large
powder volumes for a specific content of colloidal systems and thus
to a large volume of liquid composition after reconstitution. Thus,
ratios of more than about 50 or even more than 100 are less
preferred. According to the invention, ratios of less than about 50
and particularly of less than about 20 are particularly
preferred.
[0107] As pointed out, the method of the invention also allows the
preparation of liposomes in suspension and conversion into dry
particles in a single-pass procedure. The dry particles comprise a
hydrophilic excipient, which acts as a carrier for the liposomes
which are embedded or incorporated in the dry particles. The method
can be conducted at relatively low temperatures, so that it is
particularly advantageous for the drying and stabilisation of
liposomes which are thermally labile or which comprise a thermally
labile active agent in concern of spray drying.
[0108] Liposomes which have been dehydrated by the inventive method
may be re-hydrated in a suitable aqueous medium. It has
surprisingly been found that suspensions comprising liposomes
and/or lipid particles may be prepared and additionally dried by
the method of the invention in a possible one step procedure in
such a way that they can easily be reconstituted. The mild
conditions, in particular lower spray-drying temperatures using
aqueous organic solutions in combination with inert gas make the
method extremely useful for preparing and drying thermally labile
liposomes.
[0109] The invention discloses dehydrated liposomes which have been
obtained by the disclosed method. These liposomes have an average
diameter smaller than about 500 nm, preferably smaller than about
200 nm after rehydration of the liposomes in an aqueous medium,
preferably in a medium which is suitable for a pharmaceutical
application, most preferably water for injection. Liposomes may be
rehydrated by simply mixing the dehydrated liposomes with the
aqueous medium. The disclosed dehydrated liposomes have a
polydispersity index of lower than about 0.5, preferably lower than
about 0.2, most preferably lower than about 0.15, after
rehydration. The liposomes may be comprised in dry particles with
an average size between about 10 .mu.m and about 1000 .mu.m
preferably between about 50 .mu.m and about 500 .mu.m.
[0110] The liposomes, the dry particles, or a powder comprising the
same, as obtained by the method of the invention may be used in the
manufacture of a medicinal or a diagnostic preparation. If the
particles fulfil all requirements of a pharmaceutical dosage form,
it may be used as such, and filled directly into appropriate
primary packaging containers. Alternatively, the particles may be
further processed. For example, it may be mixed with further active
and/or inactive ingredients as for example pharmaceutically
acceptable carriers, or it may be compressed into a pharmaceutical
tablet.
[0111] After the reconstitution of the dry particles containing
liposomes, such as with water for injection or other buffer
systems, the resulting formulations can be used for parenteral
(e.g. i.v. or locoregional) injection, oral administration,
pulmonary or nasal inhalation. Solvents, in case they are used in
the preparation method, will be extracted during the method of
invention below permissible or detectable limits, which is a
particular advantage of the invention over methods known in the
prior art.
EXAMPLES
[0112] The following examples are meant to further illustrate the
invention and some of its preferred embodiments without limiting
its scope. Further examples and embodiments will easily be derived
from the description, optionally in combination with generally
known technical information, in particular in combination with
known principles of designing and using colloidal drug carrier
systems and spray-drying, inert spray drying or spray-freeze
drying.
Analytics of the Liposomes
[0113] The particle size (Z-average) and polydispersity (PI) of the
liposomes were analyzed by photon correlation spectroscopy (PCS)
using a Malvern Zetasizer Nano (Malvern Instruments; UK). The lipid
concentration of DOTAP-Cland DOPC was analyzed by HPLC using an
UVNIS detector at 205 nm. Separation and quantification of the
components was carried out using a C8 LiChrospher 60 RP-selected B
column (250.times.4 mm, 5 .mu.m particle size) with a C18
pre-column.
Liposome Preparation
[0114] Cationic liposomes with total lipid content between 10 to
400 mM were prepared by ethanol injection in the aqueous organic
phase. DOTAP-Cl (1,2-dioleoyl-3-trimethylammonium-propane-chloride)
and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), both from
Avanti Polar Lipids (Alabaster, USA), were dissolved in ethanol.
Multilamellar liposomes formed spontaneously upon the injection
into 10.5% trehalose solution (Ferro Pfanstiehl, USA) up to an
organic content of 30%. The suspensions were passed through the
polycarbonate membranes of 100 or 200 nm pore size and nozzle with
an orifice diameter of 0.1 and 0.2 once through in order to obtain
liposomes with defined size distribution.
Analytics of Particles
[0115] Residual moisture was determined by a coulometric Karl
Fischer titrator with a head-space oven (Analytic Jena AG,
Germany). Particle size distribution was measured with a He--Ne
laser beam equipped laser diffraction analyzer (Mastersizer X,
Malvern, Germany).
Example 1
[0116] Cationic liposomes with total lipid content of 10 mM were
prepared by ethanol injection as dislosed for example by Mundus et
al in WO 2004/002468. DOTAP-Cl
(1,2-dioleoyl-3-trimethylammonium-propane-chloride) 5 mM and DOPC 5
mM (1,2-dioleoyl-sn-glycero-3-phosphocholine), both from Avanti
Polar Lipids (Alabaster, USA), were dissolved in ethanol.
Multilamellar liposomes formed spontaneously upon the injection
into 10.5% trehalose solution (Ferro Pfanstiehl, USA).
[0117] The aqueous-organic multilamellar liposome dispersion as
prepared by ethanol injection was passed through a porous device
connected to a orifice nozzle using an ISCO DM 260 syringe pump.
The applied flow rates and resulting pressures are indicated in
Table 1. The porous device consisted of a polycarbonate membrane
(Osmonics Inc., USA) having a pore size of 200 nm supported from
both sides by a polyester drain disk (Osmonics Inc., USA) followed
by a stainless steel frit having a filtration porosity of 5 .mu.m
further supporting the membrane and drain an the membrane/drain
disk side facing the connected nozzle. A 121 VG 1/4 nozzle
(Schlick, Germany) with an orifice diameter of 0.1 mm was used.
Liposomes were collected after passing the nozzle and analysed.
[0118] The properties of liposomes prepared by the described method
with differing lipid concentrations, flow rates and pressures are
shown in Table 1.
TABLE-US-00001 TABLE 1 Lipid Flow Ethanol Average concentration
rate Pressure concentration diameter Polydispersity [mM] [ml/min]
[bar] [w/v] [nm] index 10 5 3.1 2.5 153.5 .+-. 7.7 0.23 .+-. 0.01
10 10 6.1 2.5 150.0 .+-. 5.5 0.23 .+-. 0.02 10 40 45.2 2.5 144.5
.+-. 4.9 0.19 .+-. 0.01 10 60 138.0 2.5 130.0 .+-. 2.8 0.19 .+-.
0.01 10 80 338.0 2.5 134.0 0.17 10 100 378.5 2.5 127.5 .+-. 7.7
0.14 .+-. 0.02 40 20 150.0 2.5 175.5 0.24 80 20 170.2 2.5 180.5
0.25
Example 2
[0119] The method was conducted in accordance with example 1, using
a polycarbonate membrane having a pore size of 100 nm passing. The
properties of liposomes prepared by the described method with
different flow rates and pressures are shown in Table 2.
TABLE-US-00002 TABLE 2 Lipid Ethanol Average concentration Flow
rate Pressure concentration diameter Polydispersity [mM] [ml/min]
[bar] [w/v] [nm] index 10 5 4.6 2.5 144.5 .+-. 2.1 0.26 .+-. 0.01
10 10 7.7 2.5 146.5 .+-. 4.9 0.19 .+-. 0.05 10 40 49.6 2.5 133.5
.+-. 3.5 0.18 .+-. 0.01 10 60 200.1 2.5 123.0 .+-. 2.8 0.16 .+-.
0.05 10 80 344.9 2.5 119.5 .+-. 2.1 0.17 .+-. 0.02
Example 3
[0120] The method was conducted in accordance with example 1, using
a nozzle having an orifice of 0.2 mm. The properties of liposomes
prepared by the described method with differing lipid
concentrations, flow rates and pressures are shown in Table 3.
TABLE-US-00003 TABLE 3 Lipid Flow Ethanol Average concentration
rate Pressure concentration diameter Polydispersity [mM] [ml/min]
[bar] [w/v] [nm] index 10 5 2.6 2.5 162.5 .+-. 12.0 0.25 .+-. 0.02
10 10 5.4 2.5 152.0 .+-. 12.7 0.24 .+-. 0.01 10 40 20.0 2.5 137.5
.+-. 7.7 0.18 .+-. 0.01 10 60 62.1 2.5 124.5 .+-. 210.6 0.17 .+-.
0.01 10 80 65.0 2.5 119.0 0.17
Example 4
[0121] The method was conducted in accordance with example 1 expect
that the ethanol concentration of the aqueous-organic dispersion
was varied between 10% and 30% [w/v]. The properties of liposomes
prepared by the described method with differing ethanol
concentrations, flow rates and pressures are shown in Table 4.
TABLE-US-00004 TABLE 4 Lipid Flow Ethanol concentration rate
Pressure concentration Average Polydispersity [mM] [ml/min] [bar]
[w/v] diameter [nm] index 10 5 1.0 10 220 .+-. 43.1 0.47 .+-. 0.18
10 15 8.5 10 168 .+-. 61.5 0.46 .+-. 0.06 10 60 396.0 10 80 0.29 10
5 0.8 20 144 .+-. 9.8 0.46 .+-. 0.05 10 15 6.0 20 98 .+-. 3.5 0.35
.+-. 0.06 10 30 23.2 20 70 .+-. 2.1 0.29 .+-. 0.01 10 60 296.0 20
59 0.30 10 5 1.5 30 114 .+-. 23.3 0.28 .+-. 0.01 10 15 7.5 30 75
.+-. 24.7 0.13 .+-. 0.01 10 30 22.1 30 50 .+-. 6.3 0.16 .+-. 0.05
10 60 115.0 30 43 0.09
Example 5
[0122] The method was conducted in accordance to example 4 expect
that a polycarbonate membrane having a pore size of 200 nm was
employed. The properties of liposomes prepared by the described
method with differing ethanol concentrations, flow rates and
pressures are shown in Table 5.
TABLE-US-00005 TABLE 5 Lipid Flow Ethanol concentration rate
Pressure concentration Average Polydispersity [mM] [ml/min] [bar]
[w/v] diameter [nm] index 10 5 1.7 10 118 .+-. 2.8 0.24 .+-. 0.03
10 30 13.5 10 106 .+-. 2.1 0.21 .+-. 0.02 10 60 440.0 10 82 0.20 10
5 2.5 20 84 .+-. 3.5 0.21 .+-. 0.05 10 15 14.5 20 84 .+-. 0.7 0.16
.+-. 0.02 10 30 59.7 20 71 .+-. 3.5 0.16 .+-. 0.05 10 60 309.2 20
60 0.28 10 5 1.7 30 71 .+-. 0.7 0.12 .+-. 0.02 10 15 8.1 30 69 .+-.
4.9 0.10 .+-. 0.04 10 30 30.2 30 52 .+-. 2.8 0.09 .+-. 0.00 10 60
118 30 42 0.07
Example 6
[0123] The method was conducted in analogy to example 1 except that
the aqueous-organic dispersion was pumped through the polycarbonate
membrane having a pore size of 200 nm passing the nozzle having an
orifice of 0.1 mm or 0.2 mm diameter the nozzle at a 20 ml/min flow
rate with a ethanol concentration of 10 or 30% [w/v]. The resulting
atomisation pressure was between 20 and 22 bar.
[0124] After atomisation the dispersion into droplets by passing
the dispersion through the nozzle (4), the moisture was evaporated
by contacting the droplets with the inert drying gas in a drying
tower (5) (B-290 and B-295, Buchi, Switzerland). The resulting dry
particles were separated from the inert gas by a cyclone (Buchi,
Switzerland). The drying inert gas temperature was heated to an
inlet temperature of 200.degree. C. using the heater (feature of
Buchi B290), resulting in an outlet temperature of about 75.degree.
C. The inert drying gas, gaseous nitrogen was supplied with a
constant drying volumetric flow rate of 38 m.sup.3/hour. The
separated solid and smooth particles had a particle size in the
range of 50 to 500 .mu.m with a residual moisture below 3%. After
reconstitution with water for injection a liposomal dispersion was
obtained. The resulting average diameter and polydispersity index
of the liposomes is shown in Table 6. The set-up of the equipment
is depicted in FIG. 1.
TABLE-US-00006 TABLE 6 Nozzle Inlet Outlet Lipid Flow Ethanol
Average Diameter Temp. Temp. conc. rate Pressure conc. diameter
Polydispersity [mm] [.degree. C.] [.degree. C.] [mM] [ml/min] [bar]
[w/v] [nm] index 0.1 200 75 10 20 22 10 138 0.14 0.1 200 78 10 20
20 30 162 0.42 0.2 200 87 10 20 8 10 144 0.17 0.2 200 87 10 20 7 20
179 0.33
Example 7
[0125] After the formation of liposomes by injection of an
ethanolic lipid solution into a trehalose solution as described in
Example 1, ethanol, or methanol, or acetone were added to the
liposome suspension to yield a final concentration of the solvents
of 40% [w/v]. The resulting dispersion was pumped through a
polycarbonate membrane with a pore size of 200 nm with a flow rate
of 20 ml/min and subsequently passed through a 0.1 mm orifice
nozzle as described before.
[0126] After atomizing the dispersion into droplets, said droplets
were dehydrated as set out in Experiment 6 except that an inlet
temperature of 120.degree. C. was used. Properties of the
re-hydrated liposomes were analysed as described before. The
resulting outlet temperatures, average size and polydispersity
index are shown in Table 7.
[0127] By the use of acetone as a co-solvent a very low drying
temperature of only 45.degree. C. could by achieved. Notably the
use of acetone also yielded a low polydispersity index of 0.13.
Thus the use of acetone as a co-solvent in the described method
seems to be beneficial especially for the preparation of liposomes
comprising temperature sensitive active agents.
TABLE-US-00007 TABLE 7 Nozzle Inlet Outlet Flow Ethanol Average
Poly- Diameter Temp. Term. Co- rate concentration diameter
dispersity [mm] [.degree. C.] [.degree. C.] solvent [ml/min] [w/v]
[nm] index 0.1 120 52 ethanol 20 10 83 0.44 0.1 120 55 methanol 20
30 103 0.45 0.1 120 45 acetone 20 10 125 0.13
FIGURE LEGEND
[0128] FIG. 1: Apparatus for the preparation and dehydration of
liposome in a single device. [0129] (1) Syringe pump [0130] (2)
Porous device [0131] (3) Inlet temperature [0132] (4) Nozzle [0133]
(5) Drying tower [0134] (6) Outlet temperature [0135] (7) Cyclone
[0136] (8) Product [0137] (9) Inert loop (optionally)
[0138] FIG. 2: Apparatus for the preparation of liposomes in a
single-pass mode. [0139] (10) Stock solution [0140] (11) pump
[0141] (12) filter holder [0142] (13) drain disc [0143] (14)
membrane, preferably polycarbonate membrane [0144] (15) porous
material, preferably a frit [0145] (16) nozzle.
* * * * *